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- EMDB-26816: Kinetically trapped misfolded state of the Tetrahymena ribozyme -

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Basic information

Entry
Database: EMDB / ID: EMD-26816
TitleKinetically trapped misfolded state of the Tetrahymena ribozyme
Map dataMap of misfolded state (M state) of Tetrahymena ribozyme sharpened with a B factor of 167. Refinement was performed in cryoSPARC using a 'non-uniform refinement' job.
Sample
  • Complex: apo L-21 ScaI Tetrahymena Ribozyme RNA
    • RNA: RNA (386-MER)
Keywordsribozyme / catalytic RNA / folding intermediate / misfolded / kinetic trap / M state / Tetrahymena / RNA
Biological speciesTetrahymena thermophila (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsBonilla SL / Vicens Q / Kieft JS
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118070 United States
Howard Hughes Medical Institute (HHMI)Hanna H. Gray Postdoctoral Fellowship United States
CitationJournal: Sci Adv / Year: 2022
Title: Cryo-EM reveals an entangled kinetic trap in the folding of a catalytic RNA.
Authors: Steve L Bonilla / Quentin Vicens / Jeffrey S Kieft /
Abstract: Functional RNAs fold through complex pathways that can contain misfolded "kinetic traps." A complete model of RNA folding requires understanding the formation of these misfolded states, but they are ...Functional RNAs fold through complex pathways that can contain misfolded "kinetic traps." A complete model of RNA folding requires understanding the formation of these misfolded states, but they are difficult to characterize because of their transient and potentially conformationally dynamic nature. We used cryo-electron microscopy (cryo-EM) to visualize a long-lived misfolded state in the folding pathway of the group I intron, a paradigmatic RNA structure-function model system. The structure revealed how this state forms native-like secondary structure and tertiary contacts but contains two incorrectly crossed strands, consistent with a previous model. This incorrect topology mispositions a critical catalytic domain and cannot be resolved locally as extensive refolding is required. This work provides a structural framework for interpreting decades of biochemical and functional studies and demonstrates the power of cryo-EM for the exploration of RNA folding pathways.
History
DepositionMay 2, 2022-
Header (metadata) releaseAug 31, 2022-
Map releaseAug 31, 2022-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26816.png

Downloads & links

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Map

FileDownload / File: emd_26816.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of misfolded state (M state) of Tetrahymena ribozyme sharpened with a B factor of 167. Refinement was performed in cryoSPARC using a 'non-uniform refinement' job.
Voxel sizeX=Y=Z: 1.01156 Å
Density
Contour LevelBy AUTHOR: 0.075
Minimum - Maximum-0.85168564 - 1.234832
Average (Standard dev.)0.0007139583 (±0.020021368)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 258.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened map of misfolded state (M state) of...

Fileemd_26816_additional_1.map
AnnotationUnsharpened map of misfolded state (M state) of Tetrahymena ribozyme. Refinement was performed in cryoSPARC using a 'non-uniform refinement' job.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B of misfolded state (M state)...

Fileemd_26816_half_map_1.map
AnnotationHalf map B of misfolded state (M state) of Tetrahymena ribozyme sharpened with a B factor of 167. Refinement was performed in cryoSPARC using a 'non-uniform refinement' job.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A of misfolded state (M state)...

Fileemd_26816_half_map_2.map
AnnotationHalf map A of misfolded state (M state) of Tetrahymena ribozyme sharpened with a B factor of 167. Refinement was performed in cryoSPARC using a 'non-uniform refinement' job.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : apo L-21 ScaI Tetrahymena Ribozyme RNA

EntireName: apo L-21 ScaI Tetrahymena Ribozyme RNA
Components
  • Complex: apo L-21 ScaI Tetrahymena Ribozyme RNA
    • RNA: RNA (386-MER)

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Supramolecule #1: apo L-21 ScaI Tetrahymena Ribozyme RNA

SupramoleculeName: apo L-21 ScaI Tetrahymena Ribozyme RNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Misfolded state.
Source (natural)Organism: Tetrahymena thermophila (eukaryote)

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Macromolecule #1: RNA (386-MER)

MacromoleculeName: RNA (386-MER) / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Molecular weightTheoretical: 125.096773 KDa
SequenceString: GGAGGGAAAA GUUAUCAGGC AUGCACCUGG UAGCUAGUCU UUAAACCAAU AGAUUGCAUC GGUUUAAAAG GCAAGACCGU CAAAUUGCG GGAAAGGGGU CAACAGCCGU UCAGUACCAA GUCUCAGGGG AAACUUUGAG AUGGCCUUGC AAAGGGUAUG G UAAUAAGC ...String:
GGAGGGAAAA GUUAUCAGGC AUGCACCUGG UAGCUAGUCU UUAAACCAAU AGAUUGCAUC GGUUUAAAAG GCAAGACCGU CAAAUUGCG GGAAAGGGGU CAACAGCCGU UCAGUACCAA GUCUCAGGGG AAACUUUGAG AUGGCCUUGC AAAGGGUAUG G UAAUAAGC UGACGGACAU GGUCCUAACC ACGCAGCCAA GUCCUAAGUC AACAGAUCUU CUGUUGAUAU GGAUGCAGUU CA CAGACUA AAUGUCGGUC GGGGAAGAUG UAUUCUUCUC AUAAGAUAUA GUCGGACCUC UCCUUAAUGG GAGCUAGCGG AUG AAGUGA UGCAACACUG GAGCCGCUGG GAACUAAUUU GUAUGCGAAA GUAUAUUGAU UAGUUUUGGA G

GENBANK: GENBANK: X54512.1

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7
Details: Buffer contained 50 mM NaMOPS, pH 7.0 and 10 mM MgCl2
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 32.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 98071
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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